The general organization of structural genes and transcriptional sites of heterogeneous nuclear RNA (HnRNA) in man will be studied with the use of our new high-resolution prophase chromosome technique and an improved in situ hybridization method that allows the simultaneous visualization of a radioactive probe and G-banded chromosomes. Sequences to be hybridized include 125I-labeled polysomal poly(A)-rich mRNA from HeLa cells for detection of multiple and single copy genes; 125I-labeled double-stranded regions of HnRNA isolated from HeLa cells; 3H-cRNA to HeLa cell inverted repeat DNA, believed to code for double stranded regions of HnRNA; 3H-cRNA to two cloned sea urchin histone gene segments, one of which contains the H1, H2B and H4 genes and the other the H2A and H3 genes; and mouse Beta-globin-Lambda recombinant DNA, labeled with 3H by nick-translation. In addition, total mRNA and Beta-globin and histone gene sequences will be localized on chimpanzee and gorilla phophase chromosomes. Based on preliminary evidence from our laboratory, we have proposed that messenger RNA and high molecular weight pre-messenger RNA molecules are transcribed primarily from G-negative bands of metaphase and prophase chromosomes. Confirmation and extension of these findings should broaden considerably our understanding of how the main transcriptional units of the human genome are organized, refine the present resolution of gene mapping from a regional to band-subband level, and provide insight into the pathophysiology of chromosome defects in man.